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Table 1
Hydrogenated Castor Oil
Hold Time
Initial Tempera- Final at Final
Tempera- ture Tempera- Tempera-
ture Ramp ture ture Total Time
(°) (°/min) (°) (min) (min)
80 0 80 1 1

80 30 140 0 3

140 20 180 5 10

180 2 250 10 55

Carrier gas: Hydrogen

Flow rate: 5.0 mL/min, constant flow mode
Injection volume: 1.0 µL
Injection type: Split ratio, 120:1 or 60:1
Liner: Single taper, low-pressure drop liner with
deactivated wool
Run time: 55 min
System suitability
Sample: Standard solution 1
[NOTE—See Table 2 for relative retention times.]

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CAS RN®: 8001-78-3.
DEFINITION Table 2
Hydrogenated Castor Oil is refined, bleached, hydrogenated, Component Relative Retention Time
and deodorized Castor Oil, consisting mainly of NLT 70.0%
of the triglyceride of hydroxystearic acid. ci Methyl palmitate (C16:0) 0.27

Methyl stearate (C18:0) 0.37

IDENTIFICATION
• A. IDENTITY BY FATTY ACID COMPOSITION Methyl arachidate (C20:0) 0.54
Diluent: Chloroform
Methyl 12-ketostearate 0.85
Standard solution 1: 0.8 mg/mL each of methyl palmitate,
methyl arachidate, and methyl 12-ketostearate, 8 mg/mL Methyl 12-hydroxystearate 1.00
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of methyl stearate, and 0.4 mg/mL of methyl
12-hydroxystearate, from USP Methyl Palmitate RS, methyl Suitability requirements
arachidate, methyl 12-ketostearate, USP Methyl Stearate Resolution: NLT 5 between any two adjacent peaks
RS, and USP Methyl 12-Hydroxystearate RS, in Diluent Relative standard deviation: NMT 2.0% for the peak
Standard solution 2: 4 mg/mL each of methyl stearate and area ratio of methyl 12-hydroxystearate to methyl
methyl 12-hydroxystearate, from USP Methyl Stearate RS
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12-ketostearate
and USP Methyl 12-Hydroxystearate RS, in Diluent Analysis
Sample solution: Transfer 140 mg of Hydrogenated Castor Samples: Standard solution 1, Standard solution 2, and
Oil to a 10-mL screw-cap test tube, add 3.0 mL of Diluent, Sample solution
and mix well. Add 0.5 mL of 0.5 M sodium methoxide in Calculate the relative response factor, F, for methyl
methanol,1 and mix with the sample. Allow the reaction to 12-hydroxystearate:
proceed at room temperature for 2 h. After 2 h, add 5 mL
of water, and mix. Centrifuge the test tube at 1000 × g for F = (r S/r R) × (C R/C S)
5–15 min at 4° until a clear organic layer forms. Separate
the organic layer, and remove the aqueous layer. Place an rS = peak area of methyl stearate from Standard
aliquot of the organic layer into an autosampler vial. solution 2
Chromatographic system rR = peak area of methyl 12-hydroxystearate from
(See Chromatography á621ñ, System Suitability.) Standard solution 2
Mode: GC CR = concentration of USP Methyl 12-Hydroxystearate
Detector: Flame ionization RS in Standard solution 2 (mg/mL)
Column: 0.32-mm × 30-m fused silica capillary bonded CS = concentration of USP Methyl Stearate RS in
with a 0.25-µm layer of phase G16 Standard solution 2 (mg/mL)
Temperatures
Injection port: 240° Correct the peak area of methyl 12-hydroxystearate in the
Detector: 250° Sample solution by multiplying by F.
Column: See Table 1 for oven program. Calculate the percentage of each fatty acid component in
the portion of sample taken:

Result = (r U/r T) × 100

rU = peak area of each individual fatty acid methyl

ester, except for the uncorrected peak area of
methyl 12-hydroxystearate (or the corrected
peak area of methyl 12-hydroxystearate), from
1 0.5M sodium methoxide in methanol is available from Sigma-Aldrich the Sample solution
(www.sigmaaldrich.com), product #403067. Any other equivalent reagent
can be used as well.

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rT = sum of all the peak areas, excluding the solvent Table 5

and methyl 12-hydroxystearate peaks and Relative
including the corrected peak area of methyl Retention
12-hydroxystearate, from the Sample solution Component Time
Glyceryl tri(12-hydroxystearate)
Acceptance criteria: Hydrogenated Castor Oil exhibits the [or tri(12-hydroxystearoyl)-glycerol] 1.0
composition profile of fatty acids shown in Table 3.
Di(12-hydroxystearoyl)-(12-oxostearoyl)-glycerol 1.1
Table 3 Di(12-hydroxystearoyl)-palmitoyl-glycerol 1.7
Percentage Di(12-hydroxystearoyl)-stearoyl-glycerol 1.8
Component (%)
Di(12-hydroxystearoyl)-arachidoyl-glycerol 1.9
Palmitic acid (C16:0) ≤2.0

Stearic acid (C18:0) 7.0–14.0 Suitability requirements

Arachidic acid (C20:0) ≤1.0 Resolution: NLT 1.5 between glyceryl
tri(12-hydroxystearate) and
12-Ketostearic acid (or 12-oxostearic acid) ≤5.0
di(12-hydroxystearoyl)-(12-oxostearoyl)-glycerol
12-Hydroxystearic acid 78.0–91.0 Tailing factor: 0.8–1.8 for the glyceryl
tri(12-hydroxystearate) peak
Any other unspecified fatty acid or impurity ≤3.0
Relative standard deviation: NMT 2% for the peak area
of glyceryl tri(12-hydroxystearate)
• B. MELTING RANGE OR TEMPERATURE á741ñ, Procedures, Analysis

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Procedure for Class II : 85°–88° Samples: System suitability solution and Sample solution
Calculate the percentage of each of the triglycerides in the
ASSAY
portion of sample taken:
• TRIGLYCERIDE COMPOSITION
Solution A: Methanol Result = (r U/r T) × 100
Solution B: 2-Propanol
Mobile phase: See Table 4.

Table 4
ci rU

rT
= peak area of each individual triglyceride from the
Sample solution
= sum of all the peak areas, excluding the solvent
Time Solution A Solution B peak, from the Sample solution
(min) (%) (%)
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0 100 0 Acceptance criteria: Hydrogenated Castor Oil exhibits the
composition profile shown in Table 6.
20 50 50

23 0 100 Table 6
Percentage
25 100 0
Component (%)
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35 100 0
Glyceryl tri(12-hydroxystearate)
[or tri(12-hydroxystearoyl)-glycerol] ≥70.0
Diluent: Methylene chloride Di(12-hydroxystearoyl)-(12-oxostearoyl)-glycerol ≤14.0
System suitability solution: 3.0 mg/mL of USP
Hydrogenated Castor Oil RS in Diluent. [NOTE—Due to low Di(12-hydroxystearoyl)-palmitoyl-glycerol ≤2.0
solubility, sonicate the solution for about 2–3 min.] Di(12-hydroxystearoyl)-stearoyl-glycerol 10.0–23.0
Sample solution: 3.0 mg/mL of Hydrogenated Castor Oil in
Diluent. [NOTE—Due to low solubility, sonicate the solution Di(12-hydroxystearoyl)-arachidoyl-glycerol ≤2.0
for about 2–3 min.]
Chromatographic system IMPURITIES
(See Chromatography á621ñ, System Suitability.) • LIMIT OF NICKEL
Mode: LC [CAUTION—When using closed high-pressure digestion
Detector: Evaporative light-scattering vessels and laboratory microwave equipment, the
Column: 4.6-mm × 25-cm; 5-µm packing L1 safety precautions and operating instructions given
Temperatures by the manufacturer must be followed.]
Column: 25° [NOTE—If an alternative apparatus is used, adjustment
Detector: 40° of the instrument parameters may be necessary.]
Flow rate: 1.0 mL/min Nickel standard stock solution: Dilute nickel standard
Injection volume: 5 µL solution TS two-fold with water. This solution contains the
Run time: 35 min equivalent of 5 µg/mL of nickel.
[NOTE—Depending on the different settings of the Standard solutions: Transfer 25, 50, 75, and 100 µL of
Detector, the Temperatures and Flow rate can be Nickel standard stock solution to four identical 25-mL
adjusted as long as system suitability requirements volumetric flasks. To each flask add 0.5 mL of a 10-mg/mL
are met.] solution of magnesium nitrate, 0.5 mL of a 100-mg/mL
System suitability solution of monobasic ammonium phosphate, and 6.0 mL
Sample: System suitability solution of nickel-free nitric acid. Dilute with water to volume, and
[NOTE—See Table 5 for relative retention times.] mix well. [NOTE—Content of nickel in the nickel-free nitric
acid is NMT 0.005 ppm.] The Standard solutions contain
0.005, 0.01, 0.015, and 0.02 µg/mL of nickel, respectively.

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Sample solution: Transfer about 250 mg of Hydrogenated • ALKALINE IMPURITY

Castor Oil to a suitable high-pressure-resistant digestion Sample: 1.0 g
vessel (fluoropolymer or quartz glass), and add 6.0 mL of Analysis: Dissolve the Sample by gently heating in a mixture
nickel-free nitric acid and 2.0 mL of 30% hydrogen of 1.5 mL of alcohol and 3.0 mL of toluene. Add 0.05 mL
peroxide. Place the closed vessel in a laboratory microwave of bromophenol blue TS, and titrate with 0.01 N
oven, and digest using an appropriate program (e.g., hydrochloric acid VS to a yellow endpoint.
1000 W for 40 min). Allow the digestion vessel to cool Acceptance criteria: NMT 0.2 mL of 0.01 N
before opening. Add 2.0 mL of 30% hydrogen peroxide, hydrochloric acid.
and repeat the digestion step. Allow the digestion vessel to
cool down before opening. Quantitatively transfer to a SPECIFIC TESTS
25-mL volumetric flask, add 0.5 mL of a 10-mg/mL solution • FATS AND FIXED OILS á401ñ, Procedures, Iodine Value: NMT
of magnesium nitrate and 0.5 mL of a 100-mg/mL solution 5
of monobasic ammonium phosphate. Dilute with water to • FATS AND FIXED OILS á401ñ, Procedures,
volume, and mix well. Saponification Value: 176–182
Blank solution: Place 6.0 mL of nickel-free nitric acid and • FATS AND FIXED OILS á401ñ, Procedures, Acid Value
2.0 mL of 30% hydrogen peroxide in a suitable Sample solution: Melt 20 g in a conical flask on a steam
high-pressure-resistant digestion vessel. Prepare as bath, add 75 mL of hot alcohol that has previously been
directed in Sample solution, beginning with “Place the neutralized with 0.1 N sodium hydroxide to
closed vessel in a laboratory microwave oven, and digest phenolphthalein TS, swirl, and add 1 mL of
using an appropriate program (e.g., 1000 W for 40 min).” phenolphthalein TS.
Zero solution: In a 50-mL volumetric flask, introduce 1.0 mL Analysis: Titrate with 0.1 N sodium hydroxide VS, swirling
of a 10-mg/mL solution of magnesium nitrate, 1.0 mL of a vigorously, until the solution remains faintly pink after
100-mg/mL solution of monobasic ammonium phosphate, being shaken for 60 s.

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and 12.0 mL of nickel-free nitric acid. Dilute with water to Acceptance criteria: NMT 11.0 mL of 0.1 N sodium
volume, and mix well. hydroxide VS, corresponding to NMT 3.1 for acid value
Instrumental conditions • FATS AND FIXED OILS á401ñ, Procedures, Hydroxyl Value
(See Atomic Absorption Spectroscopy á852ñ.) Sample solution: 2 g in a glass-stoppered, 250-mL conical
Mode: Atomic absorption, equipped with a graphite
ci flask. Add 5.0 mL of a freshly prepared mixture of acetic
furnace, a background compensation system, and a anhydride and pyridine (1:3), and swirl to mix. Connect the
coated tube resistant to pyrolysis flask to a reflux condenser, and heat on a steam bath for
Analytical wavelength: 232.0 nm 2 h. Add 10 mL of water through the condenser, swirl to
Lamp: Nickel hollow-cathode mix, heat on a steam bath for an additional 10 min. The
Temperature: Maintain the drying temperature of the titration can be performed on a warm solution of 50°–65°
ffi
furnace at 120° for 35 s after a 5-s ramp; maintain the to avoid a flocculation of the substance. Add through the
ashing temperature at 1100° for 10 s after a 30-s ramp; condenser 15 mL of normal butyl alcohol that has
maintain the cooling temperature at 800° for 5 s after a previously been neutralized to phenolphthalein, remove
5-s decrease; and maintain the atomization temperature the condenser, wash the tip of the condenser and the sides
at 2600° for 7 s. [NOTE—The temperature program may of the flask with an additional 10 mL of neutralized normal
be modified to obtain optimum furnace temperatures.] butyl alcohol, and add 1 mL of phenolphthalein TS.
O

Analysis Analysis: Titrate with 0.5 N alcoholic potassium hydroxide

Samples: Standard solutions, Sample solution, and Blank VS to a faint pink endpoint. Perform a blank determination
solution on a 5.0-mL portion of the acetic anhydride–pyridine
Concomitantly determine the absorbances of the Samples mixture. To determine the amount of free acid in
using the Instrumental conditions described above. Use the Hydrogenated Castor Oil, weigh 10 g into a 250-mL conical
Zero solution to set the instrument to zero. Plot the flask, add 10 mL of pyridine that has previously been
absorbances of the Standard solutions versus the neutralized to phenolphthalein, swirl to mix, add 1 mL of
concentration, in µg/mL, of nickel, and draw the straight phenolphthalein TS, and titrate with 0.5 N alcoholic
line best fitting the plotted points. From this graph, potassium hydroxide VS to a faint pink endpoint.
determine the concentration, C T, in µg/mL, of nickel in Calculate the hydroxyl value in the portion of
Hydrogenated Castor Oil taken:
the Sample solution, and determine the concentration, C
B, in µg/mL, of nickel in the Blank solution. If necessary, Result = M r × N × [A + (B × W/D) − C]/W
dilute with the Zero solution to obtain a reading within the
calibrated absorbance range. Mr = molecular weight of potassium hydroxide, 56.1
Calculate the quantity, in µg, of nickel in each g of N = normality of the alcoholic potassium hydroxide
Hydrogenated Castor Oil taken: solution
A = volume of 0.5 N alcoholic potassium hydroxide
Result = V × (C T − C B)/W consumed by the blank (mL)
B = volume consumed in the free-acid titration (mL)
V = volume of the Sample solution and the Blank
W = weight of Hydrogenated Castor Oil taken (g)
solution, 25 mL
D = weight of Hydrogenated Castor Oil used in the
CT = concentration of nickel in the Sample solution (µg/
free-acid titration (g)
mL)
C = volume consumed in the sample titration (mL)
CB = concentration of nickel in the Blank solution
(µg/mL) Acceptance criteria: 154–163
W = weight of Hydrogenated Castor Oil taken to
prepare the Sample solution (g) ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and
Acceptance criteria: NMT 20 µg/g avoid exposure to excessive heat.

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• USP REFERENCE STANDARDS á11ñ USP Methyl Palmitate RS

USP Hydrogenated Castor Oil RS USP Methyl Stearate RS
USP Methyl 12-Hydroxystearate RS

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